A 49-residue sequence motif in the C terminus of Nav1.9 regulates trafficking of the channel to the plasma membrane.

Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

Producing amyloid fibrils in vitro: A tool for studying AL amyloidosis.

Amyloid light-chain (AL) amyloidosis is the second most common form of systemic amyloidosis which is characterized by a high level of mortality and no effective treatment to remove fibril deposition. This disorder is caused by malfunctioning of B-cells resulting in production of abnormal protein fibrils composed of immunoglobulin light chain fragments that tend to deposit on various organs and tissues. AL amyloidosis is set apart from other forms of amyloidosis in that no specific sequences have been identified in the immunoglobulin light chains that are amyloid fibril formation causative and patient specific. This unusual feature hinders the therapeutic progress and requires either direct access to patient samples (which is not always possible) or a source of in vitro produced fibrils. While isolated reports of successful AL amyloid fibril formation from various patient-specific protein sequences can be found in literature, no systematic research on this topic was performed since 1999. In the present study we have developed a generalized approach to in vitro fibril production from various types of previously reported [[1], [2], [3]] amyloidogenic immunoglobulin light chains and their fragments. We describe the procedure from selection and generation of starting material, through finding of optimal assay conditions, to applying a panel of methods to confirm successful fibril formation. Procedure details are discussed in the light of the most recent findings and theories on amyloid fibril formation. The reported protocol produces high quality AL amyloid fibrils that can subsequently be used in the development of the much-needed amyloid-targeting diagnostic and therapeutic approaches.

The R280H X-ray cross-complementing 1 germline variant induces genomic instability and cellular transformation.

X-ray repair cross complementing protein 1 (XRCC1) plays an important role in base excision DNA repair (BER) as a scaffolding protein for BER enzymes. BER is one of the basic DNA repair pathways repairing greater than 20,000 endogenous lesions per cell per day. Proper functioning of XRCC1, one of the most important players in BER, was suggested to be indispensable for effective DNA repair. Despite accumulating evidence of an important role that XRCC1 plays in maintaining genomic stability, the relationship between one of its most predominant variants, R280H (rs25489), and cancer prevalence remains ambiguous. In the current study we functionally characterized the effect of the R280H variant expression on immortal non-transformed mouse mammary epithelial C127 and human breast epithelial MCF10A cells. We found that expression of R280H results in increased focus formation in mouse C127 cells and induces cellular transformation in human MCF10A cells. Cells expressing R280H showed significantly increased levels of chromosomal aberrations and accumulate double strand breaks in the G1 cell cycle phase. Our results confirm a possible link between R280H and genomic instability and suggest that individuals carrying this mutation may be at increased risk of cancer development.

Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.

A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.